The measurement of pyridine nucleotides by enzymatic cycling.

In 1935 Negelein and Haas described a method for the determination of glucose 6-phosphate dehydrogenase activity based on measurement of the rate of reduced triphosphopyridine nucleotide formation from triphosphopyridine nucleotide (1). Since that time innumerable enzymes and substrates have been measured by the appearance or disappearance of reduced diphosphopyridine nucleotide or reduced triphosphopyridine nucleotide. In fact, with the aid of auxiliary enzymes nearly every substance of biological interest could be measured with a pyridine nucleotide system The present practical limit of sensitivity of spectrophotometric measurement of DPNH or TPNH is of the order of 1OF moles per liter or 10eg moles total.’ The present practical limit for fluorometric measurement of reduced or oxidized pyridine nucleotides is of the order of lo-* moles per liter or lo-i2 moles total. Greater sensitivity would be useful both in respect to absolute amount measurable and in respect to concentration necessary for measurement. This paper describes methods for determination of reduced or oxidized DPN or TPN at concentrations as low as 10-g M and in amounts as small as lo-l5 moles. It also indicates the possibility of extending the methods to the measurement of lo-l9 moles of either pyridine nucleotide. The coenzyme to be determined is made to catalyze an enzymatic dismutation between two substrates. After several thousand cycles one of the products is measured. Apparently, Warburg, Christian, and Griese were the first to use the cycling principle to measure TPN (3) in a system consisting of glucose-6-P and its dehydrogenase and “old yellow enzyme.” In 10 minutes, for each mole of TPN, 330 moles of O2 were consumed. Jandorf, Klemperer, and Hastings (4) used the cycling principle to measure DPN in a system containing enzymes to catalyze the conversion of fructose diphosphate to glycerol-P and P-glycerate. Each mole of DPN caused the release from bicarbonate buffer, in 1 hour, of 1300 moles of COZ. With the use of this system in the Cartesian diver, Anfinsen (5) was able to measure with precision as little as 2 x lo-l2 moles of DPN. More recently, Glock and McLean (6) measured pyri-